We propose to investigate the active site structure and the molecular mechanism of catalysis by Methane Monooxygenase (MMO) purified from the Type II methanotroph, Methylosinus trichosporium OB3b (M.t. OB3b). This enzyme catalyzes the first step in the bacterial oxidation of methane (producing methanol) and will adventitiously catalyze hydroxylation of many other saturated and unsaturated hydrocarbons. The mechanism of this 3 component enzyme system is unknown. However, studies with this enzyme as well as MMOs isolated from 2 other sources suggest that the component which actually catalyzes the hydroxylation contains none of the cofactors known to exist in other monooxygenases. Thus MMO probably uses a new mechanism for catalysis. We have purified all of the components of M.t. OB3b MMO. The system offers significant advantages over the 2 other purified MMO systems including greater stability, greater yield and a 15-fold increase in specific activity. These properties will allow the purification of gram quantities of enzyme so that biophysical techniques including optical, EPR, Mossbauer and EXAFS spectroscopies can be applied to determine the structure of the hydroxylase active site. Spectroscopy of small ligand complexes, mechanism based inhibitors, isotopically labeled substrates and inhibitors, and transient kinetics are proposed as methods to investigate the molecular mechanism. This work should yield a fundamental understanding of a new type of oxygen activation chemistry, a new role for Fe in this chemistry and perhaps new insight into the design of catalysts for oxidation of abundant hydrocarbons.